The overall goal of this application is to uncover how apoptosis is used by hematopoietic stem cells (HSC) to preserve themselves and produce myeloid cells, and to address how corruption of apoptosis regulation contributes to the development of myeloid malignancies. While a wealth of information is currently available on the mechanistic role of particular components of the apoptotic machinery, we still lack a comprehensive understanding of how apoptosis regulates the biological activity of early stem and progenitor cells. Furthermore, we still do not fully understand how corruption of apoptosis regulation endows transformed HSCs with aberrant survival properties and resistance to therapy. Here, we will use an array of complementary approaches to dissect the regulation and implication of the intrinsic mitochondrial and extrinsic death receptor (DR) pathways of apoptosis in HSC function under normal and diseased conditions. Studies in Specific Aim 1 will focus on understanding the role of the intrinsic mitochondrial pathway of apoptosis. We will extend our investigations of hematopoietic-specific BakBaxcKO mice to delineate the precise contribution of this death mechanism to blood homeostasis in vivo. We will also directly test how the ratio of Bcl2 proteins controls the balance between survival and elimination in HSCs and granulocyte/macrophage progenitors (GMP). We will perform an in vivo shRNA screen to understand the role of the pro-apoptotic Bcl2 genes and will use stabilized alpha helices of Bcl2 domains (SAHB) to probe the functional implication of the pro-survival family members. These experiments will yield a detailed molecular and cellular understanding of how apoptotic signals mediated through the intrinsic mitochondrial pathway contribute to the maintenance of a functional HSC compartment and regulate myeloid cell production. In Specific Aim 2, we will address the role of the extrinsic DR pathway of apoptosis. We will use our new in situ visualization approach to investigate how local expression of DR ligands in the BM cavity can activate the DR pathway in HSCs and GMPs. We will also use a combination of molecular profiling, ex vivo analyses and in vivo experiments in complementary genetic mouse models (i.e., Faslpr/lpr, hematopoietic-specific Caspase-8cKO, p50-/- mice) to dissect the regulation and functional outcome of DR activation in HSCs and GMPs. These experiments will provide a unique understanding of how the extrinsic DR pathway contributes to HSC maintenance and regulates myeloid cell production, either by itself or in cooperation with the intrinsic mitochondrial pathway. Studies in Specific Aim 3 will address how corruption of apoptosis regulation endows transformed HSCs with aberrant survival properties and contributes to the development of myeloproliferative neoplasms (MPN). We will use our established mouse models of human MPNs (i.e., junB-deficient and inducible tTA-BCR/ABL mice) to identify changes that occur in the regulation of the apoptotic machinery in transformed HSCs and GMPs, and to understand the functional implications of these deregulations in providing aberrant survival properties to these populations. We will also assess whether targeting these aberrant features of apoptosis regulation can be used to specifically kill transformed HSCs with leukemia-initiating stem cell (LSC) properties. These experiments will uncover how corruption of a mechanism of cell preservation normally used by HSCs to maintain blood homeostasis contributes to the aberrant function of transformed HSCs and the development of myeloid malignancies.